Connective tissues are maintained in dynamic equilibrium by the opposing effects of extracellular matrix synthesis and degradation. The extracellular connective tissue matrix consists predominantly of collagens, with proteoglycans, fibronectin, laminin and other minor components making up the remainder.
Degradation of the matrix is brought about by the release of neutral metalloproteinases from resident connective tissue cells and invading inflammatory cells that are capable of degrading at physiological pH most of the matrix macromolecules. See Table 1, below. The proteinases include the mammalian tissue collagenases, gelatinases, and proteoglycanases; leukocyte collagenase and gelatinase (Murphy et al. Biochem. J. 283: 289–221 (1982); Hibbs et al., J. Biol. Chem. 260: 2493–2500 (1985)); macrophage collagenase and elastase (Werb et al. J. 360 (1975); Banda et al., Biochem. J. 193: 589–605 (1981)); and tumour collagenases (Liotta et al., PNAS-USA 76: 2268–2272 (1979); Liotta et al., Biochem. Biophys. Res. Commun. 98: 124–198 (1981); and Salo et al., J. Biol. Chem. 258: 3058–3063 (1983)). For a general review of collagenases and their role in normal and pathological connective tissue turnover see Collagenase in Normal and Pathological Connective Tissues, David E. Woolley and John M. Evanson, eds., John Wiley & Sons Ltd. (1988).
There are over five different collagen types (I, II, III, IV, V, etc.) which are differentially distributed among tissues. There is considerable homology and structural similarity among the various collagen types. Particular collagenases show some specificity for particular collagen types. See Table 1, below; Matrisian, Trends In Genetics 6: 121–125 (1990). With regard to inhibition of collagenases and other matrix-degrading metalloproteinases, it is possible that, depending on the actual enzymes, substrates, and inhibitory mechanisms, an inhibitor could act on just one, on several, or on all collagenases and metalloproteinases.
TABLE 1MATRIX-degrading metalloproteinasesName(s)Size (kDa)DegradesRef.(1) Interstitial collagenase52 deducedI, II, III collagenScholtz et al., Cancer Res. 48: 5539–(Type I collagenase)52, 57 secreted5545 (1988)(MMP-1)PMN Collagenase75 secretedI, II, III collagenMacartney et al., Evr. J. Biochem.(MMP-8)130: 71–78 (1983).(2) 72 kDA Type IV collagenase72 secretedIV, V, VII collagen,Collier et al., J. Biol. Chem.(72 kDa gelatinase)fibronectin, gelatins263: 6579–6587 (1988)(MMP-2)92 kDa Type IV collagenase78 deducedIV, V collagen, gelatinsWithelm et al., J. Biol. Chem. 263:(92 kDa gelatinase)92 secreted17213–17221 (1989)(MMP-9)(3) Stromelysin53 deducedProteoglycans, laminin, fibronectin,Chin et al., J. Biol. Chem. 260:(transin)57, 60 secretedIII, IV, V collagen, gelatins12367–12376 (1985)(proteoglycanase)(procollagen-activiating factor)(MMP 3)Stromelysin-253 deducedIII, IV, V collagen, fibronectin,Nicholson et al., Biochemistry 28:(transin-2)gelatins5195–5203 (1989)(MMP-10)PUMP-128 deducedGelatins, fibronectinQuantin et al., Biochemistry 28:(MMP-7)28 secreted5327–5333 (1989)(Small metalloproteinase of uterus)The matrix metalloproteinases are divided into three major subclasses, indicated with arabic numerals, on the basis of their substrate specificities.The enzymes in each class are bold, and alternative names are shown in parentheses. MMP, matrix metalloproteinase; PMN, polymorphonuclear leukocyte.
The underlying basis of degradative diseases of connective tissue points to the matrix-specific metalloproteinases as having a fundamental role in the etiology of these diseases. Such diseases include dystrophic epidermolysis bullosa; rheumatoid arthritis; corneal, epidermal or gastric ulceration; peridontal disease; emphysema; bone disease; and tumor metastasis or invasion.
Most studies on connective tissue degradation and diseases involving such degradation have limited the measurement of metalloproteinases to collagenase (the most widely studied of this group of metalloproteinases). It is understood however, that the simultaneous effects of collagenase and the other matrix-degrading metalloproteinases will exacerbate the degradation of the connective tissue over that achieved by collagenase alone.
Specific natural inhibitors of collagenase were discovered in crude medium from cultured connective tissues. A metalloproteinase inhibitor known as TIMP (tissue inhibitor of metalloproteinases) has been studied with regard to physicochemical properties and the biochemistry of its interaction with collagenase, Murphy et al., J. Biochem. 195: 167–170 (1981); Cawston et al., J. Biochem. 211: 313–318 (1983); Stricklin et al., J. Biol. Chem. 258: 12252–12258 (1983), and DNA encoding it has been isolated, Docherty et al., Nature 318: 65–69 (1985); Carmichael et al., PNAS-USA 83: 2407–2411 (1986). In an in vitro cell culture model of tumor cell migration through a natural basement membrane, TIMP was able to arrest migration of a collagenase-secreting tumor cell line, Thorgeirsson et al., J. Natl. Canc. Inst. 69: 1049–1054 (1982). In vivo mouse lung colonization by murine B16-FlO melanoma cells was inhibited by injections of TIMP, Schultz et al., Cancer Research 48: 5539–5545 (1988). European Patent Publication No. EP O 189 784 also relates to TIMP.
McCartney et al., Eur. J. Biochem. 130: 79–83 (1983) reported the purification of a metalloproteinase inhibitor from human leukocytes.
DeClerck et al., Cancer Research 46: 3580–3586 (1986) described the presence of two inhibitors of collagenase in conditioned medium from bovine aortic endothelial cells.
Murray et al., J. Biol. Chem. 261: 4154–4159 (1986) reported the purification and partial amino acid sequence of a bovine cartilage-derived collagenase inhibitor.
Langley, et al. EP O 398 753 (“Metalloproteinase Inhibitor,” published Nov. 22, 1990) discloses a novel metalloproteinase inhibitor and analogs, polynucleotides encoding the same, methods of production, pharmaceutical compositions, and methods of treatment. The polypeptide of FIG. 2 therein has been referred to as TIMP-2, designating a molecule distinct from TIMP-1, supra. EP O 398 753 describes both bovine and human recombinant TIMP-2.
Staskus et al., J. Biol. Chem. 266: 449–454 (1991) reports a 21 kDa avian metalloproteinase inhibitor obtained from chicken fibroblasts. The authors note the biochemical similarities with other members of the TIMP and TIMP-2 group of proteins and state that the avian material may be a TIMP variant or may represent a third protein within the metalloproteinase inhibitor family. (This material is referred to herein as “ChIMP-3”)
Pavloff et al., J. Biol. Chem. 267: 17321–17326 (1992) discloses the cDNA and primary structure of a metalloproteinase inhibitor from chicken embryo fibroblasts.
Yang et al., PNAS-USA 89: 10676–10680 (1992) reports on the role of a 21 kDa protein chicken TIMP-3.
The present work relates to a third type of metalloproteinase inhibitor polypeptides. In one aspect, the present invention involves the cloning of recombinant human TIMP-3 nucleic acid and expression thereof.